Pyruvate is an organic chemical compound that participates in the metabolism of all cells, including prokaryotes and eukaryotes. Pyruvate is a metabolic hub, situated at the cross roads of glycolysis and mitochondrial metabolism, and is the starting metabolite for multiple cellular biosynthetic pathways. Pyruvate is a molecule of great industrial interest as is currently manufactured as dietary complement, a weight control supplement and antioxidant, also being used like starting material widely applied in chemical, pharmaceutical, and agrochemical industries. Pyruvate has antioxidant properties and is thought to modulate mitochondrial redox capacity. Pyruvate is of high biomedical interest as its metabolism is altered in pathological conditions, including diabetes, neurodegenerative conditions, and cancer.
Pyruvate is in a constant state of dynamic flux between subcellular compartments, between the cell and the extracellular space and between cells. Because the concentration of pyruvate inside compartments in the living cell cannot be determined without destroying the cell, the dynamics of pyruvate in the living body is a largely unknown area.
Normal and diseased tissues are metabolically heterogeneous, showing qualitative and quantitative differences in the expression and distribution of metabolic enzymes between neighboring cells. This suggests that there may be differences between cells in terms of metabolic concentrations and fluxes for all metabolites, and specifically in terms of pyruvate concentrations and fluxes. However, this phenomenology is currently inaccessible, as current techniques to measure pyruvate are invasive and do not have sufficient sensitivity to resolve single cells.
Standard methods to measure pyruvate are based on enzymatic reactions that are monitored by means of photometric, amperometric or other devices. Enzyme-based electrodes have been developed that can detect pyruvate with high-temporal resolution. Another approach to measure pyruvate is high performance liquid chromatography (HPLC), where pyruvate is separated from other compounds by passing the sample through a stationary phase stored in a column. Nonetheless, there is a problem in the prior art, since the existing methods are invasive as they require the extraction of samples or consume pyruvate, and therefore, they change the concentration of pyruvate in the sample. A second problem of prior art methods is their sensitivity, for they cannot detect the minute amount of pyruvate present in a single cell or a single subcellular organelle. Moreover, none of the currently available methods is capable of detecting intra-cellular or sub-cellular pyruvate no-invasively in real-time or with single cell resolution. Standard methods to measure pyruvate using enzymes are cumbersome and relatively costly as they require the production and immobilization of the enzymes on a substrate and the addition of substrates and cofactors. In this regard, prior art (Staiano et al, 2007) clearly notes the difficulties for obtaining a sensor for metabolites, including for measuring pyruvate, and it remarks: “As consequence, the development of specific sensors for biochemically relevant analytes is even more challenging. In fact, it is difficult to imagine how one would design a fluorescent probe which specifically binds pyruvate, lactate, or creatinine. Even a suitable structure could be designed and synthesized, there is no guarantee that the final molecule will display a spectral change, adequate water solubility, and a suitable affinity constant.”
The transport of pyruvate across cellular and subcellular membranes is mediated by specific membrane transporters, molecules involved in the pathogenesis of several diseases and an important target for pharmacological intervention in cancer and diabetes. There are no available methods to measure the transport of pyruvate in single cells. More specifically, current and common techniques to measure the transport of pyruvate using radioactive isotopes cannot resolve single cells and have poor temporal resolution, which hampers the study fast phenomena and of normal tissues, which are heterogeneous in their cellular composition. An existing technique is indirect and infers the transport of pyruvate in single cells from changes in pH that accompany the transport of pyruvate, but this technique is limited insofar as requires prior knowledge of the usually unknown buffering capacity of the cell and is not easily applicable in the presence of physiological bicarbonate buffers.
The rates of pyruvate production and pyruvate consumption are important parameters of cell metabolism, with relevance for hypoxia/ischemia, cancer, diabetes, mitochondrial diseases and other pathological conditions. There are no available methods to measure the rates of pyruvate production and consumption in single cells. More specifically, current and common techniques to measure the rates of pyruvate production and consumption are enzyme-based methods that cannot resolve single cells and have poor temporal resolution. Measurements using isotopes cannot resolve single cells and have poor sensitivity and temporal resolution.
Pyruvate is the main substrate for mitochondria, and the speed of pyruvate metabolism is tightly linked to the speed of cellular respiration. These are fundamental parameters of cell metabolism and are affected in several diseases including hypoxic/ischemia, cancer, diabetes and other conditions. Assessment of the speed of mitochondrial metabolism is an early step in the development of pharmaceutical drugs, which is required to rule out drug candidates that may cause adverse effects on metabolism. There are no available methods to measure the rate of pyruvate consumption by mitochondria in intact cells, in single cells or in real time. Current and common techniques for measuring the rates of mitochondrial pyruvate consumption use isotopes that cannot resolve single cells and have poor sensitivity and low temporal resolution.
An existing technique based on a genetically-encoded sensor for lactate estimates the consumption of lactate in single cells (PCT/US 12/33639 from the same Applicant, not yet published). Pyruvate and lactate are linked by the enzyme lactate dehydrogenase (LDH), which catalyzes a reaction involving NADH, NAD+ and pH. Thus, the indirect estimation of pyruvate mitochondrial consumption using lactate measurement is limited insofar as may be affected in unpredictable manner by other mechanisms affecting the activity of LDH or by the concentrations of NADH, NAD+ or by intracellular pH. Another limitation of using said lactate sensor is that lactate is also a substrate for mitochondria (Brooks, 2009), so it is not possible to ascertain with the lactate sensor how much pyruvate is being consumed and how much lactate is being consumed. Moreover, the lactate sensor may not be calibrated in cells easily, which makes quantitative measurements of lactate impractical, reason why it has been recommended that its use be only qualitative or semiquantitative (San Martin et al., 2013). On the contrary, in the first place, the sensor of the present invention provides a direct measure of pyruvate concentration, while secondly, the sensor of the present invention can be easily calibrated in non-invasive form; thus providing quantitative measurement of pyruvate concentration and pyruvate fluxes. Thirdly, the estimation of flux is not affected by unpredictable variations in LDH activity and/or NADH/NAD+ ratio or by intracellular pH.